HIV-1 encodes genes that are crucial for replication in primate cells exerting function not provided by the host. Gag, Pol, and Env products represent the main virion components, while Tat and Rev regulate intracellular transcriptional and post-transcriptional events for the controlled expression of viral genes. Of particular interest are the HIV accessory proteins Vif, Vpr, Vpu, Vpx, and Nef, which are unique to primate lentiviruses. There is now strong evidence that these proteins operate in conjunction with specific host factors. In fact, none of the HIV accessory proteins has a known enzymatic activity. Instead, these proteins function primarily if not exclusively as molecular adaptors to link viral or cellular factors to pre-existing cellular pathways. In FY15 we continued projects relating to the functional properties of SAMHD1 and its mechanism of virus restriction. Further, we continued projects related to Vif and its interaction with the host CBFb. We also continued a project involving the characterization of yet another host factor, SLFN11, and its role in virus replication and we initiated projects involving the characterization of HIV and SIV Vpr proteins and their contributions to the control of host restriction factors. BST-2/Vpu: We completed and published results from a project analyzing the functional characteristics of cysteine residues in the BST-2 ectodomain. Starting with an inactive cysteine-free monomeric form of BST-2, individual cysteine residues were reintroduced throughout the ectodomain at positions predicted to form or to not form disulfides based on the available X-ray crystal structures. Our results demonstrate significant flexibility in the positioning of cysteine residues with regard to functional BST-2 dimerization even though the propensity to catalyze dimerization generally decreased with increasing proximity of the cysteines to the C-terminus of the BST-2 ectodomain. Importantly, our data indicate that BST-2 dimerization is not sufficient for inhibition of virus release since not all dimerization-competent BST-2 variants were functional in a virus release assay. Our results expose new structural constraints governing the functional dimerization of BST-2, a property essential to its role as a restriction factor tethering viruses to the host cell. In FY15, we continued a project studying the importance of Vpu oligomerization for its various biological activities. Vpu is a small integral membrane protein, which is known to cause the degradation of the viral receptor CD4 and to enhance virus release from the cell surface. In the case of CD4, Vpu acts as a molecular adaptor to connect CD4 to an E3 ubiquitin ligase complex in the endoplasmic reticulum (ER) resulting in CD4 degradation by cellular proteasomes. This function requires signals located in Vpus cytoplasmic domain. On the other hand, Vpus TM domain is required to antagonize a cellular host factor, BST-2, which otherwise prevents virus release by tethering virus particles to the cell surface. Vpu has the propensity to form homo-oligomeric complexes capable of forming ion conducting membrane pores. However, the importance of Vpu oligomerization for its biological functions has not been experimentally addressed. One of the reasons is that to date no oligomerization-defective mutants of Vpu have been identified. However, it is known that the Vpu TM domain is a driving force in the oligomerization process. To generate oligomerization-defective Vpu mutants, we decided to use a model membrane protein whose oligomerization properties have been well-characterized and for which oligomerization-defective variants have been described. Indeed, transfer of the TM domains of this model protein conferred their oligomerization characteristics to Vpu. Vpu encoding the wt TM domain of our model protein formed dimers as well as higher-order complexes as assessed by immunoblotting whereas Vpu carrying the mutated TM domain remained monomeric. We assessed the ability of monomeric and oligomeric Vpu to induce CD4 degradation in transiently transfected 293T cells and found no significant difference. Thus, degradation of CD4 does not appear to be dependent on Vpu oligomerization. This project is ongoing and we will next assess the role of Vpu oligomerization in the regulation of virus release, the deregulation of the NFkB pathway, and other reported functions of Vpu. Vif/CBFb: In FY15, we continued a project studying the role of CBFb in the Vif-dependent control of the host restriction factor APOBEC3G (A3G). We previously reported that CBFb functions like a molecular chaperone to enhance Vif biosynthesis, to stabilize mature Vif protein, and to facilitate the assembly of an A3G-Vif-Cul5 E3 ligase complex that overall results in more efficient degradation of A3G. To further characterize these functions of CBFb, we assessed the importance of CBFb for dominant negative interference with the function of wt Vif by previously described Vif variants. Preliminary data point to a positive correlation between the ability of Vif variants to bind CBFb and their dominant-negative properties. SAMHD1/Vpx: In FY14 we continued the functional characterization of SAMHD1. SAMHD1 is a dNTPase that reduces cellular dNTP concentrations to levels too low for retroviral reverse transcription. We previously found that SAMHD1 is a phosphoprotein and that its antiviral activity but not its dNTPase activity are regulated by phosphorylation at a threonine residue located near the C-terminal end of the protein. These results suggested (a) that reducing cellular dNTP levels may be necessary but is not sufficient to inhibit retroviral infection and (b) that SAMHD1 has another intrinsic activity critical for the restriction of retroviruses. The goal of our ongoing experiments is to identify and characterize this additional function of SAMHD1. In particular, a proposed exonuclease activity of SAMHD1 is under analysis. In addition, we initiated a proteomics analysis to identify potential additional host factors involved in Vpx function SLFN11: In FY15 we continued a project involving the functional characterization of a novel host factor believed to inhibit HIV-1 replication. SLFN11 was recently reported to inhibit HIV-1 replication by depleting cells of rare tRNAs used by viruses for protein synthesis. To this end we cloned SLFN11 from two separate human cellular sources and identified a polymorphism in human SLFN11. Our results indicate that the two haplotypes of SLFN11 identified thus far differ in their functional properties with respect to their ability to interfere with HIV replication. We have created SLFN11 knockdown cell lines in the backbone of 293T for transient studies as well as in H9 cells for multi-round replication studies. These cell lines are being used to further dissect the mechanism by which SLFN11 interferes with HIV replication. Preliminary data indicate that knockdown of SLFN11 in H9 cells does not significantly impair virus replication suggesting that at least in this model system, SLFN11 does not represent a critical host restriction factor.